Novel il-17r-ecd mutants

ABSTRACT

Provided are engineered soluble hIL-17RA receptors with high affinity to hIL-17 that inhibit downstream IL17A induced signaling events in cells. Also provided are methods of inhibiting hIL-17A induced secretion of CXCL1 and/or IL-6 in cells, as well as methods of treating inflammation and/or inflammatory disorders in a subject.

FIELD

The presently described subject matter relates to mutant IL17RA-ECDpeptides, and to their use in the treatment and/or prevention ofinflammatory disorders.

BACKGROUND

Interleukin 17 (IL-17) is a family of proinflammatory cytokinesconsisting of six members (IL17A-F). hIL-17A is expressed by a uniquelineage of CD4 T cells (Th-17) and is known to stimulate fibroblasts,endothelial, epithelial cells and macrophages to produce multipleproinflammatory mediators. These mediators include IL-1, IL-6, TNF-α,NOS-2 and chemokines, resulting in the induction of inflammation(Aggarwal, S. Journal of Biological Chemistry 278, 1910-1914 (2003)).Evidence from animal studies indicates that hIL-17A and hIL-17F arecrucial for coordinating local host defense against certain bacteria andfungi in mammals. Recent studies also show that hIL-17 is activelyinvolved in a range of pathologic conditions in humans, ranging fromcommon asthma to several autoimmune diseases, such as rheumatoidarthritis, psoriasis and inflammatory bowel disease, as well asallergen-specific immune responses (Kolls, J. K. Immunity 21, 467-476(2004); Iwakura, Y. The Journal of Clinical Investigation 116, 1218-1222(2006); Nakae, S. et al. Immunity 17, 375-387 (2002)). The signaling ofhIL-17 in several cell types is mediated by binding to endogenous hIL-17receptors (hIL-17R) that contain five receptor types, IL-17RA-IL-17RE(Aggarwal, S. Journal of Leukocyte Biology 71, 1-8 (2002)). Recently itwas shown that hIL-17A signaling is mediated by a heteromeric complexcontaining, hIL-17RA and hIL-17RC chains (Toy, D. et al. The Journal ofImmunology 177, 36-39 (2006)). Both receptors are single transmembraneproteins of ˜90 kDa, and contain conserved structural motifs (Kramer, J.M. et al. The Journal of Immunology 179, 6379-6383 (2007); Gaffen, S. L.Nat Rev Immunol 9, 556-567 (2009)). The IL-17R receptors are nothomologues to any known receptors and possess unique signalingproperties, which enable Th-17 cells to act as a bridge between cells ofthe innate and adaptive immune systems (Ely, L. K., Nat Immunol 10,1245-1251 (2009)).

A crystal structure of hIL-17RA extra cellular domain (ECD) bound tohIL-17F has been recently published, revealing new insight into themolecular basis for IL-17 ligand and receptor interactions (Ely, L. K.,Nat Immunol 10, 1245-1251 (2009)). Three main interaction sites at thebinding interface were detected, Thr25-Trp31 (site 1), Leu86-Arg93 (site2) and Cys259-Arg265 (site 3), site 2 being the most prominent interfaceof the complex (Ely, L. K., Nat Immunol 10, 1245-1251 (2009)). Thestructure showed one hIL-17RA bound to the dimeric hIL-17F cytokine,leaving the second potential receptor-binding interface free to engageto a second receptor.

SUMMARY

The present inventors have engineered soluble IL17 receptors forenhanced affinity, thermostability, and biological activity relative tothe wild-type receptor. Thus, the presently described engineered solublehIL-17RA receptors are useful for treating and/or preventinginflammation as well as various inflammatory disorders that, forexample, involve hIL-17 activity.

Engineered soluble hIL-17RA receptors with high affinity to hIL-17 thatinhibit downstream IL17A induced signaling events in cells have beendeveloped. A mutant library of hIL17RA extracellular domain wasgenerated, expressed and screened for mutants with increased bindingaffinity to hIL17A relative to the WT IL17RA soluble receptor. Selectedvariants showing increased expression in E. coli cells were furtherexpressed in mammalian cells, purified and examined. Two hIL17RA-ECDvariants, V3 and V10, were found to be more thermostable and bindhIL-17A with up to 6-fold higher affinity relative to the WThIL17RA-ECD. The V3 and V10 variants contain six and five mutations,respectively which are mainly found on the protein surface distal fromthe active site. These variants were further examined using a cell basedassay for inhibition of hIL-17A induced secretion of CXCL1 and IL-6 byhuman fibroblasts. Both V3 and V10 inhibited hIL-17A induced IL-6 andCXCL1 secretion by human fibroblasts at lower concentrations than the WThIL17RA-ECD. These newly discovered IL17RA mutants are usefultherapeutic agents for the treatment of disorders that involve hIL-17Aactivity, including for example, inflammation and/or inflammatorydisorders.

The presently described subject matter is directed to an IL17RA-ECDmutant comprising or consisting of the amino acid sequence of SEQ ID NO:37.

The presently described subject matter further provides an IL17RA-ECDmutant comprising or consisting of the amino acid sequence of SEQ ID NO:37, wherein position 10 of SEQ ID NO: 37 is proline.

In addition, the presently described subject matter provides anIL17RA-ECD mutant comprising or consisting of the amino acid sequence ofSEQ ID NO: 37, wherein position 15 of SEQ ID NO: 37 is glutamic acid.

Further, the presently described subject matter provides an IL17RA-ECDmutant comprising or consisting of the amino acid sequence of SEQ ID NO:37, wherein position 44 of SEQ ID NO: 37 is asparagine.

The presently described subject matter provides an IL17RA-ECD mutantcomprising or consisting of the amino acid sequence of SEQ ID NO: 37,wherein position 45 of SEQ ID NO: 37 is valine or isoleucine.

The presently described subject matter also provides an IL17RA-ECDmutant comprising or consisting of the amino acid sequence of SEQ ID NO:37, wherein position 56 of SEQ ID NO: 37 is histidine.

The presently described subject matter provides an IL17RA-ECD mutantcomprising or consisting of the amino acid sequence of SEQ ID NO: 37,wherein position 60 of SEQ ID NO: 37 is leucine or valine.

Yet further, the presently described subject matter provides anIL17RA-ECD mutant comprising or consisting of the amino acid sequence ofSEQ ID NO: 37, wherein position 97 of SEQ ID NO: 37 is lysine.

The presently described subject matter provides an IL17RA-ECD mutantcomprising or consisting of the amino acid sequence of SEQ ID NO: 37,wherein position 109 of SEQ ID NO: 37 is lysine.

The presently described subject matter further provides an IL17RA-ECDmutant comprising or consisting of the amino acid sequence of SEQ ID NO:37, wherein position 123 of SEQ ID NO: 37 is glycine.

The presently described subject matter yet further provides anIL17RA-ECD mutant comprising or consisting of the amino acid sequence ofSEQ ID NO: 37, wherein position 156 of SEQ ID NO: 37 is aspartic acid.

The presently described subject matter provides an IL17RA-ECD mutantcomprising or consisting of the amino acid sequence of SEQ ID NO: 37,wherein position 157 of SEQ ID NO: 37 is proline.

The presently described subject matter provides an IL17RA-ECD mutantcomprising or consisting of the amino acid sequence of SEQ ID NO: 37,wherein position 240 of SEQ ID NO: 37 is serine.

The presently described subject matter also provides an IL17RA-ECDmutant comprising or consisting of the amino acid sequence of SEQ ID NO:37, wherein position 244 of SEQ ID NO: 37 is tryptophan or arginine.

The presently described subject further matter provides an IL17RA-ECDmutant comprising or consisting of the amino acid sequence of SEQ ID NO:37, wherein position 249 of SEQ ID NO: 37 is histidine.

Yet further, the presently described subject matter provides anIL17RA-ECD mutant comprising or consisting of the amino acid sequence ofSEQ ID NO: 37, wherein position 268 of SEQ ID NO: 37 is valine.

The presently described subject matter also provides an IL17RA-ECDmutant comprising or consisting of the amino acid sequence of SEQ ID NO:37, wherein position 271 of SEQ ID NO: 37 is proline.

The presently described subject matter further provides an IL17RA-ECDmutant comprising or consisting of the amino acid sequence of SEQ IDNO.:1 having at least one substitution selected from the groupconsisting of leucine at position 10 is substituted with proline,proline at position 15 is substituted with glutamic acid, aspartic acidat position 44 is substituted with asparagine, leucine at position 45 issubstituted with valine, leucine at position 45 is substituted withisoleucine, glutamine at position 56 is substituted with histidine,phenylalanine at position 60 is substituted with leucine, phenylalanineat position 60 is substituted with valine, arginine at position 97 issubstituted with lysine, arginine at position 109 is substituted withlysine, aspartic acid at position 123 is substituted with glycine,histidine at position 156 is substituted with aspartic acid, alanine atposition 157 is substituted with proline, arginine at position 240 issubstituted with serine, glycine at position 244 is substituted withtryptophan, glycine at position 244 is substituted with arginine,glutamine at position 249 is substituted with histidine, alanine atposition 268 is substituted with valine and serine at position 271 issubstituted with proline.

Yet further, the presently described subject matter provides anIL17RA-ECD mutant having at least two substitutions.

In addition, the presently described subject matter provides anIL17RA-ECD mutant having seven or less substitutions.

The presently described subject matter provides an IL17RA-ECD mutant,comprising or consisting of, the amino acid sequence of SEQ ID NO: 4 orthe amino acid sequence of SEQ ID NO: 11.

The presently described subject matter further provides an IL17RA-ECDmutant comprising or consisting of the amino acid sequence of SEQ ID NO:4.

The presently described subject matter yet further provides anIL17RA-ECD mutant comprising or consisting of the amino acid sequence ofSEQ ID NO: 11.

The presently described subject matter also provides an isolatedpeptide, comprising or consisting of the amino acid sequence of apresently described IL17RA-ECD mutant.

The presently described subject matter provides an isolated peptide,comprising or consisting of an amino acid sequence of an IL17RA-ECDmutant having at least 90% homology with the amino acid sequence of SEQID: NO 1.

Further, the presently described subject matter provides an isolatedpeptide, comprising or consisting of an amino acid sequence of anIL17RA-ECD mutant having at least 95% homology with the amino acidsequence of SEQ ID: NO 1.

Still further, the presently described subject matter provides anisolated peptide, comprising or consisting of an amino acid sequence ofan IL17RA-ECD mutant having at least 99% homology with the amino acidsequence of SEQ ID: NO 1.

The presently described subject matter provides an isolated nucleic acidmolecule encoding an IL17RA-ECD mutant according to the presentlydescribed subject matter.

The presently described subject matter also provides an expressionvector comprising the nucleic acid molecule encoding an IL17RA-ECDmutant according to the presently described subject matter.

The presently described subject matter further provides an isolated hostcell transformed or transfected with the presently described expressionvector, enabling the host cell to express the presently describedIL17RA-ECD mutant.

In addition, the presently described subject matter provides an isolatedhost cell transformed or transfected with the presently describedexpression vector, enabling the host cell to express the presentlydescribed IL17RA-ECD mutant, wherein the host cell is a mammalian cell.

Yet further, the presently described subject matter provides apharmaceutical composition, comprising or consisting of the presentlydescribed IL17RA-ECD mutant or the presently described isolated peptide;and a pharmaceutically acceptable carrier or diluent.

In addition, the presently described subject matter provides apharmaceutical composition as presently described, formulated in adosage form selected from the group consisting of an intravenous dosageform and a subcutaneous dosage form.

The presently described subject matter provides a pharmaceuticalcomposition comprising or consisting of the presently describedIL17RA-ECD mutant or the presently described isolated peptide; apharmaceutically acceptable carrier or diluent; and at least oneanti-inflammatory agent.

The presently described subject matter further provides a method oftreating inflammation or an inflammatory disorder in a subject,comprising or consisting of, administering to the subject in needthereof a therapeutically effective amount of an IL17RA-ECD mutant,pharmaceutical composition, or isolated peptide, according to thepresently described subject matter.

The presently described subject matter yet further provides a method oftreating inflammation or an inflammatory disorder in a subject, whereinadministering comprises intravenous administration or subcutaneousadministration.

The presently described subject matter also provides a method oftreating inflammation or an inflammatory disorder in a subject,comprising or consisting of, administering to the subject in needthereof a therapeutically effective amount of an IL17RA-ECD mutant,pharmaceutical composition, or isolated peptide, according to thepresently described subject matter; and administering a therapeuticallyeffective amount of at least one anti-inflammatory agent.

In addition, the presently described subject matter provides a method oftreating inflammation or an inflammatory disorder wherein the at leastone anti-inflammatory agent is selected from the group consisting of acorticosteroid, cortisol; aldosterone, hydrocortisone, hydrocortisoneacetate, cortisone acetate, tixocortol pivalate, prednisolone,methylprednisolone, prednisone, triamcinolone acetonide, triamcinolonealcohol, mometasone, amcinonide, budesonide, desonide, flucinonide,fluocinolone acetonide, halcinonide, betamethasone, betamethasone sodiumphosphate, dexamethasone, dexamethasone dodium phosphate, flucortolone,hydrocortisone-17-butyrate, hydrocortisone-17-valerate, aclometasonedipropionate, betamethasone valerate, betamethasone dipropionate,prednicarbate, clobetasone-17-butyrate, clobetasol-17-propionate,flucortolone caproate, fluocortolone pivalate, fluprednidene acetate, anon-steroidal anti-inflammatory, a cox-2 inhibitor, nimesulide,diclofenac, licofelone, aspirin, ibuprofen, naproxen, an immuneselective anti-inflammatory derivative, phenylalanine-glutamine-glycine,an herb, Harpagophytum, hyssop, ginger, turmeric, Arnica Montana, willowbark, cannabis, and any combination thereof.

Further, the presently described subject matter provides a method oftreating inflammation or an inflammatory disorder in a subject, whereinthe at least one anti-inflammatory agent is administered simultaneouslywith, before, or after administration of the IL17RA-ECD mutant, thepharmaceutical composition, or the isolated peptide.

Yet further, the presently described subject matter provides a method oftreating inflammation or an inflammatory disorder in a subject,comprising or consisting of, administering to the subject in needthereof a therapeutically effective amount of the presently describedpharmaceutical composition.

In addition, the presently described subject matter provides a method oftreating inflammation or an inflammatory disorder in a subject,comprising or consisting of administering to the subject in need thereofa therapeutically effective amount of an isolated peptide or apharmaceutical composition comprising the isolated peptide, according tothe presently described subject matter.

The presently described subject matter provides a method of inhibitinghIL-17A induced secretion of IL-6 and/or CXCL1 and/or TNF-α in a cell orpopulation of cells, comprising or consisting of administering to thecell or population of cells, an amount of the presently describedIL17RA-ECD mutant, pharmaceutical composition, or isolated peptide,effective to inhibit hIL-17A induced secretion of IL-6 and/or Gro-α.

Further, the presently described subject matter provides a method ofinhibiting hIL-17A induced secretion of IL-6 in a cell or population ofcells, comprising or consisting of administering to the cell orpopulation of cells, an amount of the presently described IL17RA-ECDmutant, pharmaceutical composition, or isolated peptide, effective toinhibit hIL-17A induced secretion of IL-6.

Yet further, the presently described subject matter provides a method ofinhibiting hIL-17A induced secretion of IL-6 and/or CXCL1, in cells of asubject, comprising or consisting of administering to the subject anamount of the presently described IL17RA-ECD mutant, pharmaceuticalcomposition, or isolated peptide, effective to inhibit hIL-17A inducedsecretion of IL-6 and/or CXCL1.

Still further, the presently described subject matter provides a methodof inhibiting hIL-17A induced secretion of IL-6 and/or CXCL1, in cellsof a subject suffering from inflammation or an inflammatory disorder,comprising or consisting of administering to the subject an amount ofthe presently described IL17RA-ECD mutant, pharmaceutical composition,or isolated peptide, effective to inhibit hIL-17A induced secretion ofIL-6 and/or CXCL1, and reduce symptoms of inflammation.

The presently described subject matter provides a method of inhibitinghIL-17A induced secretion of IL-6 and/or CXCL1, wherein the cell is amammalian cell.

The presently described subject matter also provides a method ofinhibiting hIL-17A induced secretion of IL-6 and/or CXCL1, wherein themammalian cell is a human cell.

The presently described subject matter further provides a method oftreating inflammation or an inflammatory disorder in a subject,comprising or consisting of, inhibiting hIL-17A induced secretion of oneor more of TNF-α, IL-6, and CXCL1, in cells of the subject comprisingadministering to the subject an amount of the presently describedIL17RA-ECD mutant, pharmaceutical composition, or isolated peptide,effective to inhibit hIL-17A induced secretion of one or more of TNF-α,IL-6, and CXCL1, and reduce symptoms of inflammation.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A depicts an ELISA for hIL-17RA binding to hIL-17A.

FIG. 1B depicts an ELISA for hIL-17RA specific expression in E. colicells.

FIG. 1C is a graphical representation of a hIL17RA-ECD calibrationcurve.

FIG. 2A is a Schematic representation of hIL17RA displayed on the yeastcell surface.

FIG. 2B is graphical representation of expression of hIL17RA-ECDrelative to yeast PCNA and a negative control cells using YSD.

FIG. 3A is a graphical representation of the enrichment process ofhIL17RA mutant library displayed on the yeast cell surface using FACSaccording to the fluorescent expression signal.

FIG. 3B is an additional graphical representation of the enrichmentprocess of hIL17RA mutant library displayed on the yeast cell surfaceusing FACS according to the fluorescent binding signal.

FIG. 4A is a graphical representation of the analysis of the expressionlevel of three IL17RA variants in E. coli compared to the WT hIL17RA-ECDand empty vector.

FIG. 4B is an additional graphical representation of the analysis of thebinding of three hIL17RA-ECD variants to hIL-17 using ELISA compared tothe WT hIL17RA-ECD and empty vector.

FIG. 5 is a graphical representation of an ELISA experiment for theanalysis of binding of the ten different hIL17RA-ECD variants and the WThIL17RA expressed in mammalian cells to hIL-17A immobilization on96-well plates.

FIG. 6 is a graphical representation of a temperature sensitivity ofvariants V3, V10 and hIL17RA-ECD.

FIG. 7A is a representation of the ability of WT hIL17RA-ECD to inhibithIL-17A induced secretion of CXCL1 in human fibroblasts.

FIG. 7B is a representation of the ability of WT hIL17RA-ECD to inhibithIL-17A induced secretion of IL-6 in human fibroblasts.

FIG. 8 is a representation of the ability of hIL17RA-ECD, V3 and V10variants to inhibit hIL-17A induced secretion of CXCL1 in humanfibroblasts.

FIGS. 9A and B are representations of ELISA signal of the binding ofselected hIL17RA V10 mutants to hIL17A.

FIG. 10 is a graphical representation of the levels of CXCL1 in responseto IL-17 (30 μg/mouse), and V10.

FIG. 11 is a graphical representation of the levels of CXCL1 in responseto IL-17 (30 μg/mouse), V3 and V10.

FIG. 12 is a graphical representation of mean serum concentration-timeprofiles of hIL-17R-V3 after SC dose of 15 and 30 mg/kg in C57BL/6 mice(N=3).

FIG. 13 is a graphical representation of the recovery of psoriasislesions following administration of V3 IL17R variant at differentconcentrations.

DETAILED DESCRIPTION Definitions

The below definitions serve to provide a clear and consistentunderstanding of the specification and claims, including the scope to begiven such terms.

As used herein, the phrases “active agent,” “drug product,”“pharmaceutical composition,” “pharmaceutical dosage form,” and thelike, refer to the combination of one or more active agents, forexample, one or more of the presently described IL17RA mutant peptides,and optionally one or more excipients, that is administered to a patientin need of treatment, and can be in any desired form, including forexample, in the form of a solution, an aqueous solution, an emulsion,and a suspension.

Pharmaceutical compositions containing a presently described peptide asthe active ingredient can be prepared according to conventionalpharmaceutical compounding techniques. See, for example, Remington'sPharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa.(1990). See also, Remington: The Science and Practice of Pharmacy, 21stEd., Lippincott Williams & Wilkins, Philadelphia, Pa. (2005). Suitableformulations can include, but are not limited to, injectableformulations including for example, solutions, emulsions, andsuspensions. The compositions contemplated herein may take the form ofsolutions, suspensions, emulsions, combinations thereof, or any otherpharmaceutical dosage form as would commonly be known in the art.

As used herein, the terms “administering,” “administration,” and liketerms refer to any method which, in sound medical practice, delivers acomposition containing an active agent to a subject in such a manner asto provide a therapeutic effect. One aspect of the present subjectmatter provides for oral administration of a therapeutically effectiveamount of a composition of the present subject matter to a patient inneed thereof. Other suitable routes of administration can includeparenteral, subcutaneous, intravenous, intramuscular, orintraperitoneal. The dosage administered will be dependent upon the age,health, and weight of the recipient, kind of concurrent treatment, ifany, frequency of treatment, and the nature of the effect desired.

As used herein, the term “carrier,” “excipient,” or “adjuvant” refers toany component of a pharmaceutical composition that is not the activeagent.

The term “corresponding to” is used herein to refer to similar orhomologous sequences, whether the exact position is identical ordifferent from the molecule to which the similarity or homology ismeasured. A nucleic acid or amino acid sequence alignment may includespaces. Thus, the term “corresponding to” refers to the sequencesimilarity, and not the numbering of the amino acid residues ornucleotide bases.

Each of the terms “Gro-α,” “keratinocyte-derived chemokine (KC),” and“CXCL1,” refer to a small cytokine belonging to the CXC chemokinefamily. CXCL1 is expressed by macrophages, neutrophils and epithelialcells, and has neutrophil chemoattractant activity. In humans, thisprotein is encoded by the CXCL1 gene.

The term “homology” refers to the percent of identity between twopolynucleotide or two peptide moieties. The correspondence between thesequence from one moiety to another can be determined by techniquesknown to the art. For example, homology can be determined by a directcomparison of the sequence information between two peptide molecules byaligning the sequence information and using readily available computerprograms.

In an embodiment, two amino acid sequences are “substantiallyhomologous” or “substantially similar” when at least about 50%, at least60%, at least 70%, at least 80%, at least 85%, at least 90%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99%, of the amino acids match over thedefined length of the sequences. Sequences that are substantiallyhomologous can be identified by comparing the sequences using knowntechniques.

The term “hIL-17A” as used herein, refers to a proinflammatory cytokinethat is expressed by a unique lineage of CD4 T cells (Th-17) and isknown to stimulate fibroblasts, endothelial, epithelial cells andmacrophages to produce multiple proinflammatory mediators.

The term “hIL-17RA” as used herein refers to the presently describedengineered soluble hIL-17RA receptors with high affinity to hIL-17 thatinhibit downstream IL17A induced signaling events in cells. Thepresently described engineered soluble hIL-17RA receptors exhibitincreased binding affinity to hIL17A relative to the wild type (WT)IL17RA soluble receptor. Such increased affinity of hIL-17RA to hIL17Arelative to (WT) IL17RA can be from two-fold to ten-fold, fromthree-fold to eight-fold, from three-fold to six-fold, from four-fold tosix-fold, from 5-fold to six-fold, about two-fold, about three-fold,about four-fold, about five-fold, about six-fold, about seven-fold, orabout eight-fold higher.

The term “IL17RA-ECD” as used herein, refers to the hIL-17RA extracellular domain (ECD).

As used herein, the phrase “IL17RA-ECD mutant” or “IL17RA-ECD variant”or “peptide” refers to a mutant or variant of the wild type (WT)hIL17RA-ECD (SEQ ID NO: 1) (human), peptide having at least about 90%homology to the WT IL17RA, about 91% homology, about 92% homology, about93% homology, about 94% homology, about 95% homology, about 96%homology, about 97% homology, about 98% homology, or about 99% homologyto the WT IL17RA-ECD. The presently described IL17RA-ECD mutant canexhibit a higher specific IL17RA-ECD activity towards IL-17A than thewild-type hIL17RA-ECD whose amino acid sequence is SEQ ID NO: 1. Suchincreased specific activity of IL-17RA-ECD to hIL17A relative to (WT)IL17RA-ECD can be from two-fold to ten-fold, from three-fold toeight-fold, from three-fold to six-fold, from four-fold to six-fold,from 5-fold to six-fold, about two-fold, about three-fold, aboutfour-fold, about five-fold, about six-fold, about seven-fold, or abouteight-fold, higher. Such IL17RA-ECD mutants or variants include thosehaving the amino acid sequence of SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO:4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9;SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO:14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18; SEQ IDNO: 19; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 23; SEQID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 27; SEQ ID NO: 28;SEQ ID NO: 29; SEQ ID NO: 30; SEQ ID NO: 31; SEQ ID NO: 32; SEQ ID NO:33; SEQ ID NO: 34; SEQ ID NO: 35; SEQ ID NO: 36; SEQ ID NO: 37; SEQ IDNO: 77; SEQ ID NO: 78; SEQ ID NO: 79; SEQ ID NO: 80; SEQ ID NO: 81; SEQID NO: 82; SEQ ID NO: 83; SEQ ID NO: 84; and SEQ ID NO: 85. Thedescribed IL17RA-ECD mutants are more thermostable than the WT IL17RAsoluble receptor, and thus, exhibit higher stability during long storageperiods at room temperature. For example, the presently describedmutants are stable and active at temperatures from >50° C. to 70° C.,from >50° C. to 65° C., from >50° C. to 62° C., from 55° C. to 65° C.,from 55° C. to 62° C., from >55° C. to at least 60° C., from >56° C. toat least 60° C., from >58° C. to at least 60° C., from 58° C. to 65° C.,from 58° C. to 62° C., from 59° C. to 61° C., or at about 60° C.

The term “inflammation” as used herein refers to inflammationcharacterized by a statistically significant increase in the systemicconcentration of one or more cytokines, including for example, TNF-α,CXCL1 and/or IL-6, as compared to the levels of TNF-α, CXCL1, and/orIL-6, expected in a normal, healthy population matched on the basis of,for example, one or more of age, body mass index, gender, smokingstatus, and ethnicity. For example, a median concentration of IL-6observed in a normal, healthy Caucasian population is about 1.47 pg/ml,and about 2.89 pg/ml for TNF-α.

The phrase “inflammatory disorder” as used herein refers to any diseaseor disorder characterized by inflammation, for example, involvinghIL-17A.

The term “inhibiting” or “inhibit” as used herein, refers to astatistically significant reduction in the concentration, for example,systemic concentration, of one or more cytokines including, for example,TNF-α, CXCL1, and/or IL-6, observed in response to administering thepresently described IL-17RA-ECD mutant, peptide, or pharmaceuticalcomposition, as presently described, to a cell, a population of cells,or a subject, as compared to the concentration observed in the cell,population of cells, or subject, prior to administration.

The term “isolated” as used herein designates a biological material(nucleic acid or protein) that has been removed from its originalenvironment (the environment in which it is naturally present). Forexample, a peptide present in the natural state in a plant or an animalis not isolated; however, the same peptide separated from the adjacentamino acids in which it is naturally present, is considered “isolated.”

As used herein the term “peptide” means a compound that is made up oftwo or more amino acids joined by covalent bonds which are formed by theelimination of a molecule of H₂O from the amino group of one amino acidand the carboxyl group of the next amino acid.

As used herein, the term “pharmaceutically acceptable carrier” refers toa non-toxic, inert solid, semi-solid liquid filler, diluent,encapsulating material, formulation auxiliary of any type, or simply asterile aqueous medium, such as saline. Some examples of the materialsthat can serve as pharmaceutically acceptable carriers are sugars, suchas lactose, glucose and sucrose, starches such as corn starch and potatostarch, cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose and cellulose acetate; powdered tragacanth;malt, gelatin, talc; excipients such as cocoa butter and suppositorywaxes; oils such as peanut oil, cottonseed oil, safflower oil, sesameoil, olive oil, corn oil and soybean oil; glycols, such as propyleneglycol, polyols such as glycerin, sorbitol, mannitol and polyethyleneglycol; esters such as ethyl oleate and ethyl laurate, agar; bufferingagents such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonic saline, Ringer's solution; ethyl alcoholand phosphate buffer solutions, as well as other non-toxic compatiblesubstances used in pharmaceutical formulations.

Some non-limiting examples of substances which can serve as a carrierherein include sugar, starch, cellulose and its derivatives, poweredtragacanth, malt, gelatin, talc, stearic acid, magnesium stearate,calcium sulfate, vegetable oils, polyols, alginic acid, pyrogen-freewater, isotonic saline, phosphate buffer solutions, cocoa butter(suppository base), emulsifier as well as other non-toxicpharmaceutically compatible substances used in other pharmaceuticalformulations. Wetting agents and lubricants such as sodium laurylsulfate, as well as coloring agents, flavoring agents, excipients,stabilizers, antioxidants, and preservatives may also be present.

Any non-toxic, inert, and effective carrier may be used to formulate thecompositions contemplated herein. Suitable pharmaceutically acceptablecarriers, excipients, and diluents in this regard are well known tothose of skill in the art, such as those described in The Merck Index,Thirteenth Edition, Budavari et al., Eds., Merck & Co., Inc., Rahway,N.J. (2001); the CTFA (Cosmetic, Toiletry, and Fragrance Association)International Cosmetic Ingredient Dictionary and Handbook, Tenth Edition(2004); and the “Inactive Ingredient Guide,” U.S. Food and DrugAdministration (FDA) Center for Drug Evaluation and Research (CDER)Office of Management, the contents of all of which are herebyincorporated by reference in their entirety. Examples ofpharmaceutically acceptable excipients, carriers and diluents useful inthe present compositions include distilled water, physiological saline,Ringer's solution, dextrose solution, Hank's solution, and DMSO.

These additional inactive components, as well as effective formulationsand administration procedures, are well known in the art and aredescribed in standard textbooks, such as Goodman and Gillman's: ThePharmacological Bases of Therapeutics, 8th Ed., Gilman et al. Eds.Pergamon Press (1990); Remington's Pharmaceutical Sciences, 18th Ed.,Mack Publishing Co., Easton, Pa. (1990); and Remington: The Science andPractice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins,Philadelphia, Pa., (2005), each of which is incorporated by referenceherein in its entirety.

The presently described mutants, variants or peptides may also becontained in artificially created structures such as liposomes, ISCOMS,slow-releasing particles, and other vehicles which increase thehalf-life of the peptides or polypeptides in serum. Liposomes includeemulsions, foams, micelies, insoluble monolayers, liquid crystals,phospholipid dispersions, lamellar layers and the like. Liposomes foruse with the presently described peptides are formed from standardvesicle-forming lipids which generally include neutral and negativelycharged phospholipids and a sterol, such as cholesterol. The selectionof lipids is generally determined by considerations such as liposomesize and stability in the blood. A variety of methods are available forpreparing liposomes as reviewed, for example, by Coligan, J. E. et al,Current Protocols in Protein Science, 1999, John Wiley & Sons, Inc., NewYork, and see also U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and5,019,369.

The carrier may comprise, in total, from about 0.1% to about 99.99999%by weight of the pharmaceutical compositions presented herein.

The term “purified” does not require the material to be present in aform exhibiting absolute purity, exclusive of the presence of othercompounds. Rather, it is a relative definition. A peptide is in the“purified” state after purification of the starting material or of thenatural material by at least one order of magnitude, 2 or 3, or 4 or 5orders of magnitude.

The term “substantially free of naturally-associated host cellcomponents” describes a peptide or other material which is separatedfrom the native contaminants which accompany it in its natural host cellstate. Thus, a peptide which is chemically synthesized or synthesized ina cellular system different from the host cell from which it naturallyoriginates will be free from its naturally-associated host cellcomponents.

As used herein, the term “substantially pure” describes a peptide orother material which has been separated from its native contaminants.Typically, a monomeric peptide is substantially pure when at least about60 to 75% of a sample exhibits a single peptide backbone. Minor variantsor chemical modifications typically share the same peptide sequence. Asubstantially pure peptide can comprise over about 85 to 90% of apeptide sample, and can be over 95% pure, over 97% pure, or over about99% pure. Purity can be measured on a polyacrylamide gel, withhomogeneity determined by staining. Alternatively, for certain purposeshigh resolution may be necessary and HPLC or a similar means forpurification can be used. For most purposes, a simple chromatographycolumn or polyacrylamide gel can be used to determine purity.

The term “sequence similarity” in all its grammatical forms refers tothe degree of identity or correspondence between nucleic acid or aminoacid sequences of proteins that may or may not share a commonevolutionary origin. See Reeck et al., 1987, Cell 50: 667.

The active agent is preferably administered in a “therapeuticallyeffective amount.” As used herein, the term “safe and effective amount”refers to the quantity of a component which is sufficient to yield adesired therapeutic response without undue adverse side effects (such astoxicity, irritation, or allergic response) commensurate with areasonable benefit/risk ratio when used in the presently describedmanner. The phrase “therapeutically effective amount” as used hereinrefers to an amount of the presently described active agent effective toyield a desired therapeutic response. The actual amount administered,and the rate and time-course of administration, will depend on thenature and severity of the condition being treated. Prescription oftreatment, e.g. decisions on dosage, timing, etc., is within theresponsibility of general practitioners or specialists, and typicallytakes account of the disorder to be treated, the condition of theindividual patient, the site of delivery, the method of administrationand other factors known to practitioners. Examples of techniques andprotocols can be found in Remington: The Science and Practice ofPharmacy, 21st Ed., Lippincott Williams & Wilkins, Philadelphia, Pa.,(2005).

As used herein, the terms “subject” or “individual” or “animal” or“patient” or “mammal,” refers to any subject, particularly a mammaliansubject, for whom diagnosis, prognosis, or therapy is desired, forexample, a human.

As used herein, the terms “treatment” or “treating” of a disease,disorder, or condition encompasses alleviation of at least one symptomthereof, a reduction in the severity thereof, or inhibition of theprogression thereof. Treatment need not mean that the disease, disorder,or condition is totally cured. To be an effective treatment, a usefulcomposition herein needs only to reduce the severity of a disease,disorder, or condition, reduce the severity of symptoms associatedtherewith, or provide improvement to a patient or subject's quality oflife.

As used herein, the term “prevention” of a disease, disorder, orcondition encompasses the delay, prevention, suppression, or inhibitionof the onset of a disease, disorder, or condition. The presentlydescribed peptides, including vaccine compositions, may be utilized forpurposes of preventing, suppressing or treating inflammatory disordersdescribed herein. As used in accordance with the presently describedsubject matter, the term “prevention” relates to a process ofprophylaxis in which a subject is exposed to the presently describedpeptides prior to the induction or onset of the disease/disorderprocess. This could be done where an individual has a genetic pedigreeindicating a predisposition toward occurrence of the disease/disorder tobe prevented. For example, this might be true of an individual whoseancestors show a predisposition toward certain types of, for example,inflammatory disorders. The term “suppression” is used to describe acondition wherein the disease/disorder process has already begun butobvious symptoms of the condition have yet to be realized. Thus, thecells of an individual may have the disease/disorder but no outsidesigns of the disease/disorder have yet been clinically recognized. Ineither case, the term prophylaxis can be applied to encompass bothprevention and suppression. Conversely, the term “treatment” refers tothe clinical application of active agents to combat an already existingcondition whose clinical presentation has already been realized in apatient.

Any concentration ranges, percentage range, or ratio range recitedherein are to be understood to include concentrations, percentages orratios of any integer within that range and fractions thereof, such asone tenth and one hundredth of an integer, unless otherwise indicated.

Any number range recited herein relating to any physical feature, suchas polymer subunits, size or thickness, are to be understood to includeany integer within the recited range, unless otherwise indicated.

It should be understood that the terms “a” and “an” as used above andelsewhere herein refer to “one or more” of the enumerated components. Itwill be clear to one of ordinary skill in the art that the use of thesingular includes the plural unless specifically stated otherwise.Therefore, the terms “a,” “an” and “at least one” are usedinterchangeably in this application.

Throughout the application, descriptions of various embodiments use“comprising” language; however, it will be understood by one of skill inthe art, that in some specific instances, an embodiment canalternatively be described using the language “consisting essentiallyof” or “consisting of:”

For purposes of better understanding the present teachings and in no waylimiting the scope of the teachings, unless otherwise indicated, allnumbers expressing quantities, percentages or proportions, and othernumerical values used in the specification and claims, are to beunderstood as being modified in all instances by the term “about.”Accordingly, unless indicated to the contrary, the numerical parametersset forth in the following specification and attached claims areapproximations that may vary depending upon the desired propertiessought to be obtained. At the very least, each numerical parametershould at least be construed in light of the number of reportedsignificant digits and by applying ordinary rounding techniques.

Other terms as used herein are meant to be defined by their well-knownmeanings in the art.

Methods of Producing IL17RA-ECD Mutants

The presently described peptides can be prepared by chemical synthesis,or may be manufactured using recombinant DNA technology. To prepare thepresently described peptides by chemical synthesis, publicly knownmethods may be used, for example, the presently described peptides canbe obtained by methods using azide, acid chloride, acid anhydride,compound acid anhydride, DCC, activated ester, Woodward's reagent K,carbonylimidazole, deoxidization, DCC/HONB, BOP reagent (see for exampleBozanszky, M and M. A. Ondetti, Peptide Synthesis, IntersciencePublishers, New York (1966); Schroeder and Luebke, The Peptide, AcademicPress, New York (1965); F. M. Finn and K. Hofinann, The Proteins Vol. 2,H. Nenrath, R. L. Hill ed., Academic Press Inc., New York (1976); NobuoIzumiya et al., Peptide Gosei no Kiso to Jikken (Basics and experimentsof peptide synthesis), Maruzen Co. (1985); Haruaki Yajima and ShunpeiSakakibara et al., Seikagaku Jikken Koza (Biochemical Experiment) 1,Japanese Biochemical Society ed., Tokyo Kagaku Dojin Co. (1977); ToshiyaKimura, Zoku Seikagaku Jikken Koza (Sequel to Biochemical Experiment) 2,Japanese Biochemical Society ed., Tokyo Kagaku Dojin Co. (1987)).Furthermore, the presently described peptide can be prepared by chemicalsynthesis using an automated peptide synthesizer (e.g. PE Applied BioSystems Co.). Methods, such as those described in Bulaj G., et al.(2006) Biochemistry 45, 7404, can also be used for synthesis of thepresently described peptides and refolding procedures.

Further, following the completion of reaction, the presently describedpeptides can be purified and separated by publicly known purificationmethods. For example, the presently described peptides can be purifiedand separated by a combination of solvent extraction, distillation,column chromatography, liquid chromatography, recrystallization and thelike. Where the presently described peptides obtained by the abovemethods are in a free form, publicly known methods can be used toconvert it into a salt form, and on the other hand, where the peptide isobtained in a salt form, publicly known methods can be used to convertit into a free form.

In addition, recombinant expression systems may be used to express thepresently described peptides.

Other Biological Methods

Methods involving conventional and analytical chemistry, molecularbiological and cell biological techniques are described herein. Suchtechniques are generally known in the art and are described in detail inmethodology treatises such as Classics in Total Synthesis, Targets,Strategies, Methods, K. C. Nicolaou and E. J. Sorensen, VCH, New York,1996; The Logic of Chemical Synthesis, E. J. Coney and Xue-Min Cheng,Wiley & Sons, NY, 1989; and NMR of Proteins and Nucleic Acids, Wuthrich,K., Wiley & Sons, New York, 1986. Molecular biological and cellbiological methods are described in treatises such as Molecular Cloning:A Laboratory Manual, 3rd ed., vol. 1-3, ed. Sambrook et al., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; and CurrentProtocols in Molecular Biology, ed. Ausubel et al., Greene Publishingand Wiley-Interscience, New York, 1992 (with periodic updates).

Pharmaceutical Compositions Including IL17RA-ECD Mutants Formulation

The presently described pharmaceutical compositions can be administeredsystemically or locally. They can be given in forms suitable for eachadministration route. For example, they can be administered by infusionor injection, including for example, intravenous and subcutaneous.

Details of general formulation procedures and information on additionalexcipients may be found in Remington: The Science and Practice ofPharmacy, 21st Edition.

A composition containing an effective amount of the peptides describedherein can be administered to a subject requiring treatment.

The composition of the treatment may formulated to be compatible withthe route of administration. The composition can be formulated, forexample, as a solution. See, e.g., Journal of Pharmaceutical Sciences,(1963), 52:918 et seq.

A solution for parenteral, intradermal, or subcutaneous administrationmay comprise, for example: a sterile diluent such as water, saline,glycerin, fixed oils, polyethylene glycols, propylene glycol, or othersynthetic solvents; an antibacterial agents such as benzyl alcohol ormethyl parabens; an antioxidant such as ascorbic acid or sodiumbisulfite; a chelating agent; a buffering agent such as acetate orphosphate. The solution can be stored in ampoules, disposable syringes,or plastic or glass vials.

A formulation for injection or intravenous administration can include acarrier which is a solvent or a dispersion medium. Suitable carriersinclude water, physiological saline, bacteriostatic water, Cremophor EL™(BASF, Parsippany, N.J.) phosphate buffered saline (PBS), ethanol,polyols (e.g., glycerol, glycol, propylene glycol, and the like), andmixtures thereof. These compositions must be sterile and fluid to allowinjection. Fluidity can be maintained with a coating such as lecithin ora surfactant. Microbial contamination can be prevented by the inclusionof antibacterial and antifungal agents, such as parabens, chlorobutanol,phenol, ascorbic acid, and thimerosal. Sugars and polyalcohols, such asmanitol, sorbitol, sodium chloride, can be used to maintain isotonicityin the composition.

Sterility can be insured by filter sterilization of the solution.Alternatively, the solution can be produced from components that wereindividually filter-sterilized. A filter-sterilized component can bevacuum dried or freeze dried to produce a sterile powder. Such a powdercan be rehydrated prior to injection with a sterile carrier solution.

Suitable formulations can include, for example, the presently describedmutants or variants in a vehicle of glycerol and buffer, including forexample, in 30% glycerol in 50 mM Tris, 200 mM NaCl, pH7.5.

Modes of Administration

The presently described compositions, mutants, variants and peptides,described herein can be administered, for example, by bolus injection,by continuous infusion, for example, so as to prolong contact with theepidural region or by other known methods. The compositions, mutants,variants or peptides can be infused for any amount of time. Dosage andtiming of administration can be modified according to the needs of theparticular subject, e.g., within the framework of standard clinicalprotocols for treating pain. The compositions, mutants, variants orpeptides can also be delivered by intrathecal routes, and into thebloodstream. In addition, implantable or body-mountable pumps can beused to deliver the compositions, mutants, variants or peptidesdescribed herein at a controlled rate. Alternatively, prolongedadministration can be achieved by art-known depot or sustained releaseformulations.

Dosage

An appropriate dosage for treatment must be determined. An effectiveamount of the presently described compositions, mutants, variants orpeptides is the amount or dose which is required to ameliorate a symptomin a subject. Determination of the amount or dose required to treat anindividual subject is routine to one skilled in the art, e.g., aphysician, pharmacist, or researcher.

The toxicity and therapeutic efficacy of the presently describedcomposition, mutant, variant or peptide formulations may also bedetermined. Routine protocols are available for determining the LD₅₀(the dose lethal to 50% of the population) and the ED₅₀ (the dosetherapeutically effective in 50% of the population) in non-humananimals. The therapeutic index is measured as the ratio of theLD₅₀/ED₅₀. Suitable ratios include, for example, rations greater thanabout 2, 5, 10, 50, or 100. Compounds, formulations, and methods ofadministration with high therapeutic indices can be determined, as suchtreatments have little toxicity at dosages which provide high efficacy.

In formulating a dosage range for use in humans, the effective dose ofpresently described compositions, mutants, variants or peptides can beestimated from studies with laboratory animals. A dose can be formulatedin an animal in order to achieve a desired circulating plasmaconcentration of peptide. An exemplary dose produces a plasmaconcentration which exceeds the IC₅₀ (i.e., the concentration of thetest compound which achieves a half-maximal inhibition of a symptom) asdetermined in cell culture assays. The circulating plasma concentrationcan be determined, for example, by obtaining a blood sample, and byanalyzing the sample using an antibody based specific ELISA assay orwith high performance liquid chromatography or mass spectroscopy.

Alternatively, the dose can be estimated from tests in an animal model,as described below. Alleviation of symptoms is observed when ratsreceive a peptide or pharmaceutical composition at a dose of at leastabout from 1 μg/kg to 25 mg/kg, from 1 mg/kg to 10 mg/kg, from 10 mg/kgto 25 mg/kg, or more. The interrelationship of dosages for animals andhumans (based on milligrams per meter squared of body surface) isdescribed by, for example, Freireich et al., Cancer Chemother. Rep.1966, 50, 219. Body surface area may be approximately determined fromheight and weight of the patient. See, e.g., Scientific Tables, GeigyPharmaceuticals, Ardley, N.Y., 1970, 537. See also, Principles andPractice of Pharmaceutical Medicine, Lionel D. Edwards, Andrew J.Fletcher, Anthony W. Fox and Peter D. Stonier, (2007). The presentlydescribed compositions, mutants, variants or peptides can beadministered with a frequency or continuously in order to maintain alocal concentration effective to treat an inflammatory and/or autoimmunedisorder.

Depending on the method of administration, the appropriate dose canvary. The dose for a patient can be optimized while the patient is undercare of a physician, pharmacist, or researcher. For example, arelatively low dose of the compositions, mutants, variants or peptidesdescribed can be administered initially. The patient can be monitoredfor symptoms. The dose can be increased until an appropriate response isobtained. In addition, the specific dose level for any particularsubject can vary depending on the age, body weight, general health,gender, and diet of the subject, the time of administration, the routeof administration, the rate of excretion, and other drugs provided incombination.

EXAMPLES Materials and Methods Plasmids

hIL-17RA EST clone was purchased from Open Biosystems. For E. coliexpression, the ECD of the gene was cloned into pET32 plasmid (Novagen).The cloning was performed using NcoI and NotI sites to yield aThioredoxin (Trx)-6× Histidine-tagged version of the protein. E. coliClooni strain (Lucigen) was used for cloning and plasmid extraction. Foryeast surface display, the variants were cloned into pCTCON plasmidusing the NheI and BamHI sites (Chao, G. et al. Nat. Protocols 1,755-768 (2006)). For expression in mammalian cells, the pFUSE(Invivogen), pFW02 (CrownBio) and pYD11 (CrownBio) vectors were used, toyield hIL17RA-ECD variant fused with human IgG 1 engineered Fc, withgreatly reduced antibody-dependent cellular cytotoxicity (ADCC) andcomplement dependent cytotoxicity (CDC). Primers used for the cloningand library construction are listed in Table 1.

TABLE 1 List of Primers. SEQ ID Primer name 5′ -> 3′ sequence NO.Primers for hIL17RA-ECD ECD-nested- GTGGTGGTGGTGGTGCTC 38 NotECD-nested- GTACCGACGACGACGACAAG 39 Nco fr-pCTCON-ATAAACGCTAGCTCCCTGCGACTCCTGGACC 40 ECD IL17RA ACC rev-pCTCON-TAGATGTCGGATCCGTACACCCACAGGGGCA 41 ECD IL17RA TGTAGTCC fr-pCTCON-GGTGGTGGTTCTGGTGGTGGTGGTTCTGGTG 42 ECD hrGTGGTGGTTCTGCTAGC TCCCTGCGACTCC TGGACCACC rev-pCTCON-GATCTCGAGCTATTACAAGTCCTCTTCAGAA 43 ECD hrATAAGCTTTTGTTCGGA TCCGTACACCCAC AGGGGCATGTAGTCC fr-ECDTCCCTGCGACTCCTGGACCACC 44 rev-ECD GTACACCCACAGGGGCATGTAGTCC 45 fr-pET₃₂-ATTCGATGCCATGGCCTCCCTGCGACTCCTG 46 ECD/Nco GACCACC rev-pET₃₂-TCACTCAGTGCGGCCGCCTATTAGTACACCC 47 ECD/Not ACAGGGGCATGTAGTCCGfr-ECD-Nco- CCATGGTTCTGCGACTCCTGGACCACCGGGC 48 pFC GCTG rev-ECD-TCTAGACCACAGGGGCATGTAGTCCG 49 Xba-pFC fr-ECD-GGATCCTAGAGAGGCTTGTGGGGCCTCAGG 50 Bam-pFC rev-ECD-GTCGACGCCCACAGGGGCATGTAGTCCGG 51 SalI-pFC Primers for mutants libraryL10P TGGACCACCGGGCGCCGGTCTGCTCCCAGCC 52 P15ECTGGTCTGCTCCCAGGAAGGGCTAAACTGCA 53 CG D44NCCTCCTCCCCAAAGAACCTGCAGATCCAGC 54 L45I TCCTCCCCAAAGGACATTCAGATCCAGCTGC55 AC L45V CTCCTCCCCAAAGGACGTGCAGATCCAGCT 56 GC Q56HCCCACACCCAACATGGAGACCTGTTCCC 57 F60V CAACAAGGAGACCTGGTGCCCGTGGCTCACA 58TC F60L CAACAAGGAGACCTGCTGCCCGTGGCTCACA 59 TC R97KCGTTTGTGCGTCAAATTTGAGTTTCTGTCC 60 R109K TGAGGCATCACCACAAACGGTGGCGTTTTA61 CC D123G GTGGTTGACCCTGGCCAGGAATATGAGGTG 62 H156DGTGCCTGACTGTGAGGATGCCAGGATGAAGG 63 A157P CTGACTGTGAGCATCCGAGGATGAAGGTAA64 CC R240S CACACTCACTCTAAGCAACCTTAAAGGGTG 65 G244WACGCAACCTTAAATGGTGCTGTCGCCACC 66 Q249H GTGCTGTCGCCACCATGTGCAGATCCAGC 67A268V GCCTCAGACACTCCGTGACTGTTTCCTGCCC 68 S271PCACTCCGCGACTGTTCCGTGCCCAGAAATG 69 CC Primers for sequence fr-pET₃₂-TTCCTCGACGCTAACCTGGCC 70 seq rev-pET₃₂- AGCAGCCGGATCTCAGTGGTGG 71 seqfr-pCTCON- GACGATTGAAGGTAGATACCCATACGACGTT 72 lib CC rev-pCTCON-CAGATCTCGAGCTATTACAAGTCCTCTTCAG 73 lib pFC-fr GTTTTCTGTTCTGCGCCGTTAC 74pFC-rev GCATTCTAGTTGTGGTTTGTCC 75 pFC-rev-in CATGAGGGTGTCCTTGGGTTTTGG 76

Yeast Surface Display

hIL17RA-ECD variants were displayed on the yeast cell surface of EBY100strain cells (see Chao, G. et al. (2006) for genotype) and analyzed byflow cytometry, essentially as described by Chao, G. et al. (2006).Briefly, EBY100 transformed with plasmid pCTCON containing the desiredclone were grown in SDCAA media (20 g sucrose, 6.7 g yeast nitrogenbase, 5 g casamino acids, 5.4 g Na₂HPO₄ and 8.56 g NaH₂PO₄*H₂O) tologarithmic phase, then 2*10⁶ of cells were washed, resuspended in SGCAAinduction media (similar to SDCAA but containing galactose instead ofsucrose) and grown at 37° C. with shaking for an additional 18 hours.Induced cells (1*10⁶) were collected by centrifugation, washed with PBSF(PBS+1 g/L BSA) and incubated for 1 hour at 25° C. with 0.2 μM hIL-17A(R&D Systems). The cells were then washed and incubated for 1 hour at25° C. with mouse α-Myc antibodies (Santa Cruz Biotechnology, 1 μl/50 μlPBSF) and goat biotinilated α-hIL-17A (R&D Systems, 0.25 μl/50 μl PBSF).Subsequently, cells were washed again and incubated with fluoresceinisothiocyanate (FITC) conjugated α-mouse IgG (Sigma, 1 μl/50 μl PBSF)and allophycocyanin (APC) conjugated streptavidin (JacksonImmunoresearch, 1 μl/50 μl PBSF) for an additional 1 hour on ice, withfrequent mixing. The labeled cells were washed, resuspended with PBSFand analyzed by fluorescence activated cell sorting (FACS Calibur, BD).The positive control proliferating cell nuclear antigen (PCNA) gene wasexpressed and displayed on the yeast cell surface under identicalconditions.

Mutant Library Generation

hIL17RA-ECD “back to consensus” library was generated by targetingspecific residues that deviate from the family consensus. Based on thealignment of the hIL17RA-ECD with IL17RA-ECD from other mammalians, wefound 18 positions that deviated from the consensus sequence.hIL17RA-ECD gene was amplified by PCR, and ˜10 μg were digested withDNaseI to yield 50-125 by fragments as described in Stemmer, W. P.(1994). The fragments were reassembled, as in DNA shuffling (Aharoni, A.et al. 2004) in the presence of a mixture of 18 short oligos (4-6 nMeach, Table 1), resulting in a library containing 2-6 mutations in eachgene (Stemmer, W. P. 1994). The reaction mixture was further amplifiedby nested PCR as described in Chao, G. et al. (2006). The assembledlibraries were ligated into pET32 vector for E. coli expression and intothe pCTCON vector for yeast surface display. This naïve mutationslibrary was transformed to yeast and displayed on the yeast surface(Chao, G. et al. 2006). The same library was also directly cloned intopET32 plasmid, expressed in E. coli and screened for mutants with higherexpression level than that of the original clone, as described above.

Library Selection Using Yeast Surface Display

The naïve library was induced and labeled with c-myc and hIL-17A, asdescribed above. EBY100 cells (1*10⁷) displaying the hIL-17RA librarywere labeled, analyzed and sorted using a FACS (Vantage, BD). Two tothree iterative rounds of enrichment were performed. In each round,multiple ‘positive’ events (3-5*10⁴), corresponding to cells foundwithin the top 1-2% of the green and red fluorescence intensity area,were collected into growth media and plated on agar for a new round ofenrichment. For initial sorting of the naïve library, a sort gate of thetop 5% of fluorescent cells was used. Selection rounds were continueduntil no further enrichment was obtained.

Cloning and Bacterial Expression of hIL17RA-ECD Variants

A pool of plasmids from the last round of FACS enrichment wasPCR-amplified, cloned into plasmid pET32 and transformed to E. coli BL21(DE3) strain (Novagen). Alternately, plasmids of the naïve librarieswere transformed to BL21 E. coli cells. Following transformation, singlecolonies were inoculated into 10 ml LB media containing 100 μg/mlampicillin, grown to OD₆₀₀ of 0.6 and induced with 1 mM of IPTG(Calbiochem) overnight at 30° C. The cells were then harvested, lysed inPBS supplemented with 0.2% Triton, 200 μg/ml lysozyme (Calbiochem) and10 mM β-mercaptoethanol, centrifuged and the cleared supernatant wascollected and analyzed by ELISA (see below). In each round of selection140-200 mutants were screened to identify clones with improved activityrelative to the WT protein.

Protein Expression in Mammalian Cells

For large scale purification of hIL17RA-ECD variants, HEK293F cells weretransiently transformed with 1 μg/ml of DNA dosage and DNA/PEI ratio of1:3 or 1:4, and harvested after 6-7 days. The recombinant proteins werepurified on HiTrap Protein A HP column, with 20 mM sodium phosphate pH7.0 as binding buffer and 100 mM sodium citrate pH 3.0 as elutionbuffer. The proteins were then analyzed by silver stain and western blotusing specific antibodies against the Fc. Endotoxin concentration wasalso measured. Purified proteins were stored at −80° C. in 50 mM Tris,200 mM NaCl pH 7.5 for further analysis.

ELISA Assay for hIL-17A Binding

ELISA plates (Griener Microlon 96W) were incubated with 100 μl of 0.5μg/ml goat α-hIL-17A antibodies (R&D Systems) for 1 hour, washed withPBS supplemented with 0.05% Tween-80 (PBST) and 100 μl of 0.35 μg/mlhIL-17A (R&D Systems or ProSpec Tany TechnoGene Ltd.) were added to theplate for an additional 1 hour. The plates were then washed with PBSTand blocked by incubation with 100 μl of PBS supplemented with 3% skimmilk for 1 hour. Following blocking, the plates were washed andincubated with 100 μl of the cleared lysate, cell media or purifiedprotein generated (described above) at appropriate dilutions and shakenfor 1 hour. hIL-17RA (R&D Systems) was applied at a concentration of6.25 μg/ml as a positive control, and PBS supplemented with 1% BSA as anegative control. Plates were then washed with PBST, incubated with 100μl of 0.05 μg/ml goat α-hIL-17RA antibodies (R&D Systems) followed by asecondary peroxidase-conjugated streptavidin (Jackson, 1:10000dilution). Finally, 100 μl of the horseradish peroxidase (HRP)chromogenic 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution(Dako) were added. The reaction was stopped by the addition of 1 Msulfuric acid and recorded at 450 nm using a Tecan Infinite M200 platereader.

ELISA Assay for the Detection of hIL17RA Expression in E. coli

ELISA plates (Griener Microlon 96W) were coated with 100 μl of 0.2 μgstreptavidin (Pierce) and 100 μl of 0.05 μg/ml goat α-hIL-17RAantibodies (R&D Systems). The plates were then blocked by incubationwith PBS supplemented with 3% skim milk for 1 hour. Next, 100 μl of thecleared lysates were added to each well and the plates were incubatedwith shaking for 1 hour at room temperature. After washing the plateswith PBST, the plates were incubated with 100 μl of mouse α-6×His-tagantibodies (Santa-Cruz Biotechnology, 1:2000 dilution), washed with PBSTand further incubated with secondary HRP-conjugated goat α-mouseantibodies (Jackson, 1:5000 dilution) for 1 hour. Finally, the HRPchromogenic TMB substrate solution (Dako) was added and the reaction wasstopped by the addition of 100 μL of 1M sulfuric acid and recorded at450 nm using a Tecan Infinite M200 plate reader.

Protein Thermostability

For protein thermostability tests, the purified proteins andcommercially available hIL-17RA were diluted in PBS with 1% BSA to afinal concentration of 5 μg/ml. The variants were then incubated for 30min. at different temperatures (ranging from 37 to 70° C.). The reactionwas terminated by transferring the proteins to liquid nitrogen, theproteins were kept at −80° C. and then tested for their hIL-17A bindingactivity by ELISA at room temperature.

Affinity Measurements

The affinity of hIL-17A binding to hIL17RA-ECD variants was determinedby SPR measurements on ProteOn XPR36 (Bio-Rad) instrument. All sampleswere in HBST buffer (10 mM HEPES with 0.15 M NaCl, 3.4 mM EDTA and0.005% Tween-20, pH 7.2). A GLC Chip was air initialized and activatedwith EDC/S-NHS and 4 μg from each of the hIL17RA-ECD variants werediluted in Acetate buffer pH 5.5 and immobilized onto the chip. Themeasured binding levels for WT hIL17RA-ECD, V3 and V10 were ˜5300RU,6700RU and 7400RU, respectively. Following blockage of the unbound siteson the chip with Ethanolamine, the chip was flushed with HBST buffer androtated. The hIL-17A was run at 25 μl/min. for 300 sec at variousconcentrations (50, 25, 12.5, 6.25 and 3.125 nM) followed by a 10-min.dissociation step. Binding parameters were determined using Langmuirsingle binding site model using the Bio-Rad's proteOn Manager SoftwareV2.1.2.05.

Fibroblast-Based Assay for the Inhibition of hIL17 Induced IL6 andGro-{acute over (α)} Secretion

Cell line used for the assay is the normal human skin fibroblast cellline ATCC CRL2091. Cells were routinely maintained in completed MEMmedium at 37° C. with 5% CO₂. Cell cultures for each experiment wereallowed to reach 95-100% confluence, counted and checked for viabilitywith 0.5% Trypan Blue solution (Biological Industries). Cells were thendiluted to reach 10⁵ cells/ml densities, and 100 μl of the cellsuspension are added to each well of 96 well plates (Nunc) to obtain 10⁴cells per well. The plates were incubated at 37° C. in 5% CO₂ for 24hours until 95-100% confluence was achieved, and then stimulated foradditional 24 hours with 10 ng/ml hIL-17A in presence or absence ofdifferent amounts of hIL17RA-ECD variants. Dose response of the celllines to hIL-17A was tested in each experiment by adding differentamounts of hIL-17A (0-20 ng/ml). Cell culture supernatants were thencollected and stored at −20° C. for further analysis.

Cytokine Detection

Measurement of hIL-6 and hCXCL1 in culture supernatants were performedthrough the use of ELISA kits (R&D Systems or PeproTech) according tomanufacturer description. Standard curves for IL-6 and Gro-α wereconstructed in each ELISA by adding different amounts of these cytokines(supplied with the kit) instead of the cells supernatant. PeproTechELISA kit was used with several modifications: briefly, the blockingbuffer was PBS supplemented with 3% skim milk, and PBS supplemented with1% BSA was used as a diluent. The supernatants from cells were diluted1:5 to 1:10 and used for cytokine detection. The obtained results wereconverted to pg/ml of IL-6 or CXCL1, according to the respectivestandard curves.

Example 1

To generate soluble hIL-17R with high affinity for the hIL-17A the focuswas on the extracellular domains (ECD) of the hIL-17RA receptor that isknown to bind hIL-17A. A directed evolution of hIL-17R for improvingstability and binding affinity of hIL17RA-ECD was utilized. First a“back to consensus” library of hIL17RA-ECD was generated and thendisplayed on the yeast cell surface. The library was enriched for clonesthat were able to bind hIL-17A using flow cytometry. Individual clonesfrom the enriched library were screened using ELISA and several selectedhIL-17R variants were overexpressed and purified on a large scale frommammalian cells for further characterization. Finally, pure hIL17RA-ECDvariants were tested for binding hIL-17A and inhibition of hIL-17Ainduced IL-6 and CXCL1 secretion in fibroblast cell line.

Detection of hIL-17A-hIL17-RA Interaction

An ELISA assay was developed for hIL-17RA-hIL-17 interactions in orderto facilitate the fast and sensitive screening of a large number ofhIL17RA-ECD mutants. The assay is based on immobilizing hIL-17A on ELISAplates and detection of hIL17RA-ECD binding using specific antibodies.To examine the dynamic range for detection of hIL-17RA-hIL17Ainteraction, varied amounts of the WT hIL17RA-ECD were applied to theplate. Different hIL17RA-ECD concentrations, ranging from 0.1-10 pg/ml,were examined to determine the sensitivity and dynamic range of theELISA. A solution of 0.35 μg/ml of hIL-17A was used for plate coating todetect hIL17RA-ECD binding following expression in either E. coli orHEK293T cells, and 5-10 μg/ml of the WT hIL17RA-ECD was used as apositive control (FIG. 1). The dependence of the signal on thehIL7RA-ECD concentration was linear and the assay proved to be verysensitive, enabling detection of even low receptor amounts (FIG. 1). Inaddition, an ELISA assay was developed to directly monitor thehIL17RA-ECD expression level using hIL-17R specific antibodies (FIG. 1).This ELISA assay was used in parallel with the hIL-17A binding ELISA, asan additional tool to estimate hIL17RA-ECD expression level in E. coli.

Yeast Surface Display of hIL17RA-ECD.

A yeast surface display (YSD) approach was utilized to display thehIL17RA-ECD on the yeast cell surface. Schematic representation of yeastsurface display of hIL-17RA and detection of expression and binding tohIL-17A is shown in FIG. 2A. The display level of hIL17RA-ECD wasmonitored using FITC antibody against the c-myc tag at the C-terminal ofhIL17RA-ECD. The binding to hIL-17A was monitored by biotynilatedα-hIL-17A antibody followed by streptavidin-APC antibody. It was foundthat the WT hIL17RA-ECD displayed on the yeast surface exhibits very lowlevel of expression and hIL-17A binding (FIG. 2B). Proliferating CellNuclear Antigen (PCNA) gene from Saccharomyces cerevisiae expressedunder identical conditions (Fridman, Y. et al. PLoS Biol. 2010) was usedas a positive control in these experiments (FIG. 2B).

Engineering of hIL17RA-ECD Using Directed Evolution Methodology

To improve the expression level of hIL-17R in E. coli and its affinityfor the hIL-17A ligand, a “back to consensus” library was generated bymutating residues in hIL17RA-ECD that deviate from the IL-17RA familyconsensus back to the family consensus. Based on the alignmentsummarized in Table 2, 18 relevant positions were identified thatdeviate from the consensus sequence and mutated these residues byspiking of mutated oligonucleotides to the wild type gene (Herman, A. &Tawfik, D. S. (2007). Incorporating Synthetic Oligonucleotides via GeneReassembly (ISOR): a versatile tool for generating targeted libraries.Protein Eng Des Sel 20, 219-26.). This naïve “back to consensus” librarywas transformed to yeast and displayed on the yeast surface (Chao, G. etal. Nat. Protocols 1, 755-768 (2006)).

TABLE 2 Amino Acid Residue Frequency for the hIL17RA-ECD Protein.Position^(a) hIL17RA Mutation^(c) 10 L L10P 15 P P15E 44 D D44N 45 LL45V L45I 56 Q Q56H 60 F F60L F60V 97 R R97K 109 R R109K 123 D D123G 156H H156D 157 A A157P 240 R R240S 244 G G244W 249 Q Q249H 268 A A268V 271S S271P ^(a)Amino acid residue positions are shown according to thehuman IL17RA-ECD protein sequence. ^(c)Amino acid mutations spiked intohIL17RA-ECD to generate the mutant library

To enrich the naïve library for mutants with enhanced expression andaffinity for hIL-17A, the library was analyzed and sorted byfluorescence-activated cell sorter (FACS), based on the fluorescenceexpression and binding signals. Two rounds of enrichment were performedto maintain the active clones and discard the inactive ones (FIGS. 3A,3B). Next, the enriched libraries were sub-cloned, expressed andscreened in E. coli cells for mutants showing enhanced expression levelusing ELISA. The naïve library was also directly cloned into pET32plasmid, expressed in E. coli and screened for mutants with higherexpression level than that of the WT hIL17R protein. Comparison of thetwo screening experiments clearly indicates that library enrichmentusing YSD substantially increased the number of positive clones by afactor of 5 relative to the same library directly screened in E. colicells without pre-enrichment (see Table 3).

TABLE 3 Comparison Between the Direct Screening of the Naive Library andScreening of the Same Library Following FACS Enrichment. No. of screenedNo. of improved Selection method mutants clones ELISA for naive library180 7 (~4%) ELISA for library after 2 rounds of 90 16 (~19%) FACSenrichment

All mutants were screened by both types of ELISA (FIG. 4), for thedetection of hIL-17A binding and hIL17RA-ECD expression. Overall, thefirst round of evolution yielded 22 mutants (Table 4) with higherexpression level than that of the WT hIL17RA-ECD (FIG. 4A), but withalmost no change in hIL-17A binding affinity (FIG. 4B) relative to theWT IL17RA.

These results indicate that in E. coli cells, these IL17RA mutants areexpressed at a higher level than the WT protein (FIG. 4A) but probablydue to the lack the natural post translational modification, do not bindefficiently to the IL17A ligand (FIG. 4B).

TABLE 4 List of Mutants of hIL-17RA Obtained in the First Round ofDirected Evolution and Selection. Number Number of variant Mutationsmutations V1 P15E D44N F60L R109K H156D R240S 6 V2 D44N H156D 2 V3 L10PR109K D123G H156D G244W A268V 6 V4 L45I F60L L69P H156D 4 V5 D44N F60LR97K R109K 4 V6 P15E F60L R109K D123G 4 V7 F60V R109K H156D 3 V8 P15ER109K H156D 3 V9 L10P P15E D44N F60L R97K R109K 6 V10 L10P F60V R109KD123G A157P 5 V11 P15E R109K Q124R A157P 4 V12 L10P H117R A157P 3 V13R97K H156D N194D G244W 4 V14 L45V Q56H R109K G140R H156D Q249H A268V 7V15 L45V F100I D123G A268V 4 V16 L10P P15E R109K H156D 4 V17 F60L K103ED123G H156D 4 V18 N89D D123G H156D 3 V19 R8Q P15E L45I F60L 4 V20 L10PP15E L45V F60V Q124R A157P 6 V21 F60L R109K D123G A157P E182K T204I 6V22 F60V H156D 2Expression of Selected hIL17RA Mutants from “Back to Consensus” Libraryin Mammalian Cells

Proteins expressed and purified from E. coli lack post-translationalmodifications that are available in mammalian expression systems(Durocher, Y., Perret, S. & Kamen, A. Nucleic Acids Research 30, e9(2002)), and contain high levels of LPS, a bacterial endotoxin, known toeffect mammalian cell line growth and function. The variants V1-V10 wereexpressed and purified in mammalian cells, similar to the commerciallyavailable WT hIL17RA-ECD (Table 4). The produced recombinant proteinswere highly pure (>95-98%) and contained low amounts of LPS suitable fortesting in human cells (Table 5).

TABLE 5 Analytical Summary of hIL17RA-ECD Variants Produced in MammalianCells by CrownBio. Concentration Purity by silver Endotoxin # Variant(mg/ml) stain (EU/mg) V1 1.6 >95% 3.2 V2 1.28 >98% 2.0 V3 1.3 >98% 3.4V4 0.97 >98% 5.3 V5 1.2 >98% 2.2 V6 1.58 >98% 0.8 V7 1.8 >98% 1.8 V81.5 >98% 0.4 V9 1.7 >98% 22.9 V10 1.0 >98% 2.8 ECD-FL 0.85 >98% 5.2hIL17A Binding by the hIL17RA-ECD Variants Expressed in Mammalian Cells

To characterize the ability of the different variants purified frommammalian cells to bind hIL-17A, the hIL17RA-hIL-17A interaction wasexamined by ELISA. The ten variants, V1-V10, and the WT IL17RA wereadded at three different concentrations to IL17A pre-coated plates toexamine the differences in ELISA signal (FIG. 5). It was found thatvariants V3 and V10 exhibit higher binding level than the WT hIL17RA-ECDexpressed and purified under the same conditions, or the commerciallyavailable IL17RA-ECD (FIG. 5).

To quantitatively characterize the differences between the bindingaffinities of hIL-17A to WT hIL17RA-ECD and to variants V3 and V10, therespective association and dissociation rate constants were measuredusing surface plasmon resonance (Table 6). It was found that theaffinity of the WT hIL17RA-ECD to hIL-17A is 2.6 nM that is in goodagreement with the binding affinity previously measured by Wright, J. F.et al. (Wright, J. F. et al. The Journal of Immunology 181, 2799-2805(2008)). Variants V3 and V10 show 6 and 4 fold increased affinity(K_(d)) relative to the WT hIL17RA-ECD, respectively

TABLE 6 Kinetic Rate constants of hIL-17A Cytokine Binding to WThIL17RA-ECD, V3 and V10 Variants as Determined by SPR. Fold increaseImmobilized in ligand k_(a) (1/M * s) k_(d) (1/s) K_(d) (M) affinity WThIL17RA- 1.54 × 10⁵ 4.04 × 10⁻⁴ 2.62 × 10⁻⁹ ± 0.04 — ECD V3 1.70 × 10⁵7.51 × 10⁻⁵ 4.43 × 10⁻¹⁰ ± 0.2 6 V10 1.84 × 10⁵ 1.15 × 10⁻⁴ 6.24 × 10⁻¹⁰± 0.15 4.2

Temperature Sensitivity of the V3 and V10 Variants

To further characterize the thermostability of the V3 and V10 variantsrelative to the WT hIL17RA-ECD, the different proteins were incubatedfor 30 min. at different temperatures, ranging from 48° C. to 70° C. Theheat inactivated samples were then tested using the ELISA to measure theresidual binding activity of each variant to hIL-17A. It was found thatthe WT IL17RA exhibits no loss in binding signal following incubation attemperatures between 48° C.-50° C. (FIG. 6). However, followingincubation of the WT hIL17RA-ECD at ˜60° C. a significantly lowerresidual binding to hIL-17A was observed relative to the same sampleincubated at room temperature. In contrast, it was found that both V3and V10 variants were highly active following incubation at 60° C.,indicating a shift of ˜5° C. in their heat inactivated temperaturecompared to the WT hIL17RA-ECD (FIG. 6). Specifically, FIG. 6 shows thethermal inactivation of WT IL-17RA (triangle), V3 (filled circle) andV10 (empty circle) mutants. The IL-17RA variants were incubated atdifferent temperatures for 30 min., and residual IL-17RA binding toIL-17A was monitored at room temperature using ELISA. The results werenormalized relative to the variant binding following incubation at 50°C. The heat inactivation temperatures derived from the fits are57.1±0.6, 63.8±1.2 and 63.9±0.5 for the WT, V3 and V10, respectively.Each data point is the average of two independent experiments.

These results show that the V3 and V10 variants are more thermostablethan the WT IL17RA soluble receptor, and thus, exhibit higher stabilityduring long storage periods at room temperature.

Cell Based Assay for IL-6 and Gro-α Secretion

To examine the ability of the recombinant hIL-17RA variants to inhibithIL-17A binding to fibroblasts, a cell based assay was established. Theassay is based on measuring Gro-α and IL-6 secretion following theaddition of hIL-17A to the fibroblast cells. The addition of solublehIL17RA-ECD together with the hIL-17A prevents its binding to theendogenous hIL-17RA receptor, thus leading to reduction in IL-6 andGro-α secretion. It was found that addition of 0.5 μg/ml commercialhIL17RA-ECD to the fibroblasts together with 10 ng/ml hIL-17A issufficient to inhibit both Gro-α and IL-6 secretion (FIGS. 7A and 7Brespectively). Inhibition of Gro-α was consistent with the inhibition ofIL-6 indicating the efficient inhibition of hIL-17A binding to theendogenous receptor.

Next, the level of inhibition of IL17 induced secretion of Gro-αfollowing the addition of V3 and V10 variants relative to the soluble WTIL17RA were examined. Both mutants were able to inhibit Gro-α secretionin human fibroblasts at much lower concentrations than the WT protein(FIG. 8). It was found that a significantly lower level of Gro-α issecreted in presence of V3 and V10 at concentrations of 0.25 μg/ml or0.1 μg/ml relative to the hIL17RA-ECD at the same concentrations.

These results show that the V3 and V10 variants are much more potent ininhibiting the IL17A induced secretion of Gro-a and IL6, and thus, areuseful for treating and/or preventing IL17 induced inflammatorydisorders.

Example 2 Screening of V10 Random Mutant Library

Error-Prone Library Generation.

Libraries were performed with mutagenic dNTP analogs, 8-oxodG and dPTP,as previously described (Aharoni et al., 2004, Harel et al, 2004).

Variant V10 isolated from the “back to consensus” library was used as atemplate for error-prone PCR in order to generate a random mutantlibrary. Individual plasmids containing the V10 mutated gene wereisolated from 350 individual colonies following growth in liquid media(Table 7). More than three hundred V10 mutants were then individuallytransfected, expressed in mammalian cells, and tested for binding tohIL17A using the ELISA for the detection of IL17-IL17R interaction.Eight clones (V23-V30) exhibited similar or higher hIL17A binding thanthe wt ECD and the original V10 clone expressed under identicalconditions (FIG. 9A).

TABLE 7 List of Mutants of V10. # of # variant Mutations mutations V23F60V I75T R109K D123G A157P M209T 6 V24 F60V E79K R109K D123G A157PI201V 6 V25 H53Y F60V R109K D123G A157P G244R 6 V26 N18S F60V L76P R109KD123G A157P 6 V27 V21I F60V R109K D123G A157P 5 V28 N89A 1 V29 N89AF190I 2 V30 F60V R109K D123G H131Y A157P M209R 6 V31 H53Y F60V R109KD123G A157P 5 V32 F60V E79K R109K D123G A157P 5 V33 N18S F60V N89A R109KD123G A157P 6 V34 H33Y F60V R109K D123G A157P I201V 6 V35 F60V E79KR109K D123G F113L A157P 6

Next DNA shuffling between IL17R V23-V30 genes (FIG. 9A) was preformedto generate new IL17R library. More than one hundred individual mutantsfrom the DNA shuffling library were expressed in mammalian cells andanalyzed for binding to hIL-17A using the ELISA. Five variants (V31-V35)exhibited higher binding to hIL17A relative to the WT IL17RA ECD or theV10 clone expressed under identical conditions (FIG. 9B).

Example 3 Screening of V10 and V3 Mutant Library

The variants V10N1-V10N8 and V3N2 as presented in table 8 are derivedfrom V10 and V3 respectively with the addition of mutations that wereidentified in V23-V35 (Table 7) that are beneficial for the stabilityand or binding and or activity in cell based assay. All these variantsare currently generated by CrowBio and expressed in HEK293 to obtainpure protein for subsequent analysis using ELISA and cell based assay.

TABLE 8 List of Mutants of V10 and V3 # of # variant Mutations mutationsV10N1 L10P F60V R109K D123G A157P N89A 6 V10N2 L10P F60V R109K D123GA157P E79K 6 V10N3 L10P F60V R109K D123G A157P H53Y 6 V10N4 L10P F60VR109K D123G A157P N18S 6 V10N5 L10P F60V R109K D123G A157P L76P 6 V10N6L10P F60V R109K D123G A157P I201V 6 V10N7 L10P F60V R109K D123G A157PM209T 6 V10N8 L10P F60V R109K D123G A157P M209R 6 V3N2 N89A R109K D123GH156D 4

Example 4 The Effect of V3 and V10 on Cytokines Levels

To mimic the effect of IL-17 secreted in, for example, an autoimmuneinflammatory disease, IL-17 was injected with or without wt-ECD or oneof the ECD-mutants into mice and the levels of various cytokines weremeasured.

IL-17 (30 μg/mouse) was injected into mice and blood samples for CXCL1levels assay were collected 2 hrs after the injection.

FIG. 10 shows that the levels of CXCL1 cytokine are increased as aresult of IL-17 (30 μg/mouse) administration via injection while V10 (14mg/Kg) mutant did not increase CXCL1 (KC) levels. Furthermore,preincubation of IL-17 with V10 (14 mg/Kg) prior to injection decreasedthe secreted CXCL1 level by about 40%.

In FIG. 11 shows that the levels of CXCL1 (KC) cytokine are increased asa result of IL-17 (30 μg/mouse) administration via injection preincubation of the IL-17 with the wt form of the IL-17R (ECD) or themutants V3(14 mg/Kg) or V10(14 mg/Kg) inhibit the secreted levels ofCXCL1 (KC) cytokine.

The most potent form is the V3(14 mg/Kg) inhibits CXCL1 levels by morethan 80%. The wt-ECD and V10 (14 mg/Kg) inhibit secreted CXCL1 by 60%and 70%, respectively.

Example 5 Pharmacokinetic Analysis of V3 Variant in Mice

A group of 24 male C57BL/6 mice with a weight of 24-26 g was used forthe study. The mice were divided to two groups of 12 mice and theh-IL17R-V3 was administered at concentrations of 15 mg/kg or 30 mg/kgvia subcutaneous injection (Table 9). The mice had free access to waterand food. Blood from the mice was collected at ten different time pointsfollowing infection. The blood collection was performed followinganesthetized under Isoflurane. Approximately 110 μL blood/time point wascollected into tube via retro-orbital or cardiac puncture. Blood samplewas centrifuged to obtain serum sample (2000 g, 5 min. under 4° C.) atroom temperature within 30 minutes. The serum samples were stored at−70° C. until analysis by using ELISA. Stability of the V3 in the miceserum was very high with a half life time (T_(1/2)) of up to 99.4 hours(Tables 10 and 11). In addition, a high concentration of V3 of up to4.85 mg/mL in the mice sera was observed (FIG. 12).

The results show high stability of V3 in the sera and the potential ofobtaining high concentration of V3 in the sera with few injections overlong periods of time.

TABLE 9 Study design of pharmacokinetic study (PK) Dose Dose DoseTreatment No. of Route of Level Conc. Volume Group Treatment animalsadmin. (mg/kg) (mg/mL) (mL/kg) Time points 1 hIL-17R-V3 12 SC 15 4.03.75 0.5, 1, 2, 4, 8, 24, 48, 72, 96 & 120 hr, serum collection only 2hIL-17R-V3 12 SC 30 4.0 7.50 0.5, 1, 2, 4, 8, 24, 48, 72, 96 & 120 hr,serum collection only

TABLE 10 PK results Individual and mean serum concentration-time data ofhIL-17R-V3 after SC dose of 15 mg/kg in C57BL/6 mice^(a,b) SamplingConcentration Mean Dose Dose time (μg/mL) (μg/ CV (mg/kg) Route (hr)Individual mL) SD (%) 15 SC 0.5 BQL BQL BQL NA NA NA 1 4.35 2.94 4.904.06 1.01 25.0 2 BQL 2.43 2.35 2.39 0.0540 2.26 4 3.38 3.02 4.13 3.510.569 16.2 8 2.16 3.18 3.06 2.80 0.559 19.9 24 4.11 3.80 3.70 3.87 0.2135.49 48 2.98 2.86 2.99 2.94 0.076 2.59 72 2.33 2.80 3.19 2.77 0.429 15.596 BQL BQL BQL NA NA NA 120 BQL BQL BQL NA NA NA PK parameters UnitEstimated Value Tmax hr 1.00 Cmax μg/mL 4.06 T_(1/2) hr 99.4 AUC_(last)hr * μg/mL 228 AUC_(INF) hr * μg/mL 625 ^(a)BQL: Below QuantifiableLimit of 1.95 μg/mL for hIL-17R-V3 in mouse serum sample ^(b)NA: NotAvailable

TABLE 11 PK results Individual and mean serum concentration-time data ofhIL-17R-V3 after SC dose of 30 mg/kg in C57BL/6 mice^(a,b) SamplingConcentration Mean Dose Dose time (μg/mL) (μg/ CV (mg/kg) route (hr)Individual mL) SD (%) 30 SC 0.5 2.74 BQL *11.1 2.74 NA NA 1 BQL BQL BQLNA NA NA 2 7.81 2.84 3.89 4.85 2.62 54.0 4 3.03 5.32 4.76 4.37 1.19 27.38 3.55 3.74 4.68 3.99 0.604 15.1 24 4.76 4.60 4.82 4.73 0.112 2.37 483.70 4.27 3.53 3.83 0.386 10.1 72 3.24 3.8 3.77 3.63 0.344 9.49 96 2.482.40 1.99 2.29 0.263 11.5 120 2.05 BQL BQL 2.05 NA NA PK parameters UnitEstimated Value Tmax hr 2.00 Cmax μg/mL 4.85 T_(1/2) hr 76.1 AUC_(last)hr * μg/mL 417 AUC_(INF) hr * μg/mL 642 ^(a)BQL: Below QuantifiableLimit of 1.95 μg/mL for hIL-17R-V3 in mouse serum sample ^(b)NA: NotAvailable

Example 6 Contribution of the Different Mutations to IL-17A Activity andThermostability

To examine the contribution of the back to consensus mutationsidentified in V3 and V10 to the activity and thermostability of theproteins, a series of site directed mutants were generated and examined.The majority of the mutations found in V3 and V10 individually weremutated back to the WT residue. These mutants were expressed inmammalian cells to maintain posttranslational modifications and examinedfor binding to IL-17A using ELISA.

The thermostability of the active mutants following 30 min. ofincubation at elevated temperatures was examined and characterized bytheir residual activity using ELISA. Detailed analysis of single mutantsof the V3 indicated that two back-to consensus mutations, D123G andH156D were completely essential for IL17-RA binding to IL-17A (Table12). The results suggest that R109K mutation bring about 50% decrease inbinding to IL-17A but significant decrease in thermostability. Thisanalysis also showed that the two mutations, G244W and A268V, that werenot part of the back-to consensus mutations and were randomlyincorporated during the PCR amplification steps, only slightlycontribute to the V3 activity or stability. Similar analysis ofmutations in V10M confirmed the importance of D123G and R109K for IL-17Abinding and thermostability, respectively. The V10 analysis alsorevealed that F60V mutation is crucial for IL-17A binding. Themutational analysis indicates that most of the back-to consensusmutations are essential to IL-17RA binding and indicates that thesemutations cooperatively contribute to IL-17RA activity. However, thisanalysis indicated that it was possible to reduce the number ofmutations by back mutation of L10P, G244W and A268V thus reducing thenumber of mutations in V3 from six to three mutations enabling to lowerthe risk of immunogenicity following administration.

TABLE 12 Mutational analysis of V3 and V10 IL-17RA variants IL-17A^(c)binding IL-17RA relative variant^(a) Mutations^(b) to the WT i (°C.)^(d) V3 L10P, R109K, D123G, H156D, 100% 62 G244W, A268V V3 P10LR109K, D123G, H156D, 85% 62 G244W, A268V V3 K109R L10P, D123G, H156D,G244W, 45% 58 A268V V3 G123D L10P, R109K, H156D, G244W, <5% ND A268V V3D156H L10P, R109K, D123G, G244W, <5% ND A268V V3 W244G L10P, R109K,D123G, H156D, 65% 62 A268V V3M V268A L10P, R109K, D123G, H156D, 80% 62G244W V10 L10P, F60V, R109K, D123G, A157P 100% 62 V10 V60F L10P, R109K,D123G, A157P 10% ND V10 K109R L10P, F60V, D123G, A157P 80% 58 V10 G123DL10P, F60V, R109K, A157P <5% ND ^(a)IL-17RA variants were expressed inmammalian cells. The back-to-WT mutations in V3 and V10 are highlightedin bold. ^(b)List of mutations in each variant following the differentback-to-WT mutations ^(c)Binding of the UL-17RA mutant was measured byELISA ^(d)Temperature of inactivation determined by the residual bindinglevel following incubation at various temperatures ND—Not determined.

Example 7 In Vivo Analysis of V3 in Psoriasis Mice Models

To assess the therapeutic potential of V3 variant in the recovery ofpsoriasis lesions, the therapeutic effect of V3 in mice model containingpsoriasis lesions was examined. Skin samples from psoriasis patientswere transplanted into mice and 28 days following skin transplant V3 wasadministered via subcutaneous injection twice a week for 4 weeks.Different concentrations of V3 including 3.5 mg/kg, 7 mg/kg, 14 mg/kgand 28 mg/kg were examined. After 56 days from the initial skintransplant the mice were sacrificed and the recovery of the psoriasislesions was assessed using histology analysis. The level of recovery wasscored from 1 to 4 where 1 is sick, 2 is partial recovery, 3 issignificant recovery and 4 is a complete recovery (Table 13). As can beseen below (FIG. 13 and Table 13), disease was present in 6/8 of themice treated with PBS. Treatment with Dexamethasone inhibited disease in6/8 mice, similar to historical data within this model. sV3R was givento mice at 4 different doses, where the 7 mg/kg significantly inhibiteddisease in 6/8 mice similar to the effect observed with dexamethasone.Attenuated response at higher V3 concentration was found, probably dueto protein aggregation prior administration (FIG. 13).

TABLE 13 Summary of testing of V3 IL17RA in psoriasis mice model Partialto Partial Complete Complete Psoriasis Recovery Recovery Recovery Avg PValue Score 1 Score 2 Score 3 Score 4 Score (vs PBS) PBS 5/8 1/8 2/8   2± 0.5 Dexa 2/8 6/8 3.25 ± 0.5  0.077 V3 3.5 mg/kg 4/8 4/8 2.5 ± 0.60.473 V3 7 mg/kg 1/8 1/8 6/8 3.4 ± 0.4 0.043 V3 14 mg/kg 4/8 4/8 2.5 ±0.6 0.473 V3 28 mg/kg 3/8 1/8 4/8 2.6 ± 0.5 0.326

It will be appreciated by those skilled in the art to which the presentsubject matter pertains that various modifications can be made withoutdeparting from the essential nature thereof. It is intended to encompassall such modification within the scope of the appended claims.

1. A protein, comprising the amino acid sequence of SEQ ID NO:
 37. 2.The protein of claim 1, wherein position 10 of SEQ ID NO:37 is proline,position 15 of SEQ ID NO:37 is glutamic acid, position 18 of SEQ IDNO:37 is serine, position 44 of SEQ ID NO:37 is asparagines, position 45of SEQ ID NO:37 is valine or isoleucine, position 53 of SEQ ID NO:37 istyrosine, position 56 of SEQ ID NO:37 is Histidine, position 60 of SEQID NO:37 is Leucine or valine, position 76 of SEQ ID NO:37 is proline,position 79 of SEQ ID NO:37 is lysine, position 89 of SEQ ID NO:37 isalanine, position 97 of SEQ ID NO:37 is lysine, position 109 of SEQ IDNO:37 is lysine, position 123 of SEQ ID NO: 37 is glycine, position 156of SEQ ID NO:37 is aspartic acid, position 157 of SEQ ID NO:37 isproline, position 201 of SEQ ID NO:37 is valine, position 209 of SEQ IDNO:37 is threonine or arginine, position 240 of SEQ ID NO:37 is serine,position 244 of SEQ ID NO:37 is tryptophan or arginine, position 249 ofSEQ ID NO:37 is Histidine, position 268 of SEQ ID NO:37 is valine,position 271 of SEQ ID NO:37 is proline, or any combination thereof.3-24. (canceled)
 25. A protein comprising the amino acid sequence of SEQID NO.:1 having at least one substitution selected from the groupconsisting of leucine at position 10 is substituted with proline,proline at position 15 is substituted with glutamic acid, asparagine atposition 18 is substituted with serine, aspartic acid at position 44 issubstituted with asparagine, leucine at position 45 is substituted withvaline, leucine at position 45 is substituted with isoleucine, histidineat position 53 is substituted with tyrosine, glutamine at position 56 issubstituted with histidine, phenylalanine at position 60 is substitutedwith leucine, phenylalanine at position 60 is substituted with valine,leucine at position 76 is substituted with proline, glutamic acid atposition 79 is substituted with lysine, asparagine at position 89 issubstituted with to alanine, arginine at position 97 is substituted withlysine, arginine at position 109 is substituted with lysine, asparticacid at position 123 is substituted with glycine, histidine at position156 is substituted with aspartic acid, alanine at position 157 issubstituted with proline, isoleucine at position 201 is substituted withvaline, methionine at position 209 is substituted with threonine,methionine at position 209 is substituted with arginine, arginine atposition 240 is substituted with serine, glycine at position 244 issubstituted with tryptophan, glycine at position 244 is substituted witharginine, glutamine at position 249 is substituted with histidine,alanine at position 268 is substituted with valine and serine atposition 271 is substituted with proline, and combination thereof.26-27. (canceled)
 28. A protein, comprising the amino acid sequence ofSEQ ID NO:4 or the amino acid sequence of SEQ ID NO:11. 29-34.(canceled)
 35. An isolated nucleic acid molecule encoding the protein ofclaim
 1. 36. An expression vector comprising the nucleic acid of claim35.
 37. A cell transformed or transfected with the expression vector ofclaim
 36. 38. The cell of claim 37, wherein said cell is a mammaliancell.
 39. A pharmaceutical composition, comprising the protein accordingto claim 1; and a pharmaceutically acceptable carrier or diluent. 40.The pharmaceutical composition of claim 39, formulated in a dosage formselected from the group consisting of an intravenous dosage form and asubcutaneous dosage form.
 41. The pharmaceutical composition of claim39, further comprising at least one anti-inflammatory agent.
 42. Amethod of treating an inflammatory disorder in a subject, comprisingadministering to the subject in need thereof a therapeutically effectiveamount of the protein according to claim
 1. 43. The method of claim 42,wherein administering comprises intravenous administration orsubcutaneous administration.
 44. The method of claim 42, whereinadministering further comprises administering a therapeuticallyeffective amount of at least one antiinflammatory agent. 45-46.(canceled)
 47. A method of treating an inflammatory disorder in asubject, comprising administering to the subject in need thereof atherapeutically effective amount of the pharmaceutical composition ofclaim
 39. 48. The method of claim 47, wherein administering comprisesintravenous administration or subcutaneous administration.
 49. A methodof inhibiting hIL-17A induced secretion of one or more of CXCL1 and IL-6in a cell, comprising administering to the cell an amount of theIL17RA-ECD mutant according to claim 1, in an amount effective toinhibit hIL-17A induced secretion of one or more of TNF-a IL-6 andCXCL1-.
 50. The method of claim 49, wherein the cell is a mammaliancell.
 51. The method of claim 50, wherein the mammalian cell is a humancell.
 52. A method of treating inflammation in a subject, comprisinginhibiting hIL-17A induced secretion of one or more of IL-6, CXCL1, andTNF-a, in cells of the subject comprising administering to the subjectan effective amount of the protein of claim 1.